随州和安陆银杏古树种核变异的分子遗传基础
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摘要
银杏(Ginkgo biloba L.)属银杏科银杏属,为现存种子植物中最古老的孑遗植物之一,有“活化石”之称。我国作为世界银杏的起源地,银杏古树资源十分丰富,湖北省的随州和安陆是主要分布地之一。本研究在前人研究随州和安陆银杏古树种核特征和种仁营养成分的基础上(付慧敏,2006),进一步从分子水平对其变异状况进行研究。主要研究结果如下:
(1)用9条ISSR引物对120个银杏单株种核胚乳的基因组DNA进行PCR扩增,共得到145条清晰的扩增谱带。其中,多态性谱带116条,多态位点百分率(PPB)为80.00%。在总体水平上,Nei's基因多样性H_E为0.269,Shannon's多态性信息指数H_o为0.401,表明随州和安陆银杏种核在总体水平上具有较广泛的遗传变异。
(2)用多态位点百分率(PPB)、观测等位基因数(A_o)、有效等位基因数(A_E)、Nei's基因多样性值(H_E)以及Shannon's多态性信息指数(H_o)来度量四种种核类型内的遗传变异,结果表明:不同种核类型遗传变异程度依次是梅核类＞马铃类＞圆子类＞佛指类。四种种核类型间的遗传分化值G_(ST)为0.228,φ_(ST)=2.46%,说明银杏古树的遗传变异主要分布在类型内。
(3)根据单株间的遗传相似系数(GS),应用UPGMA法构建120个银杏单株的分子聚类图;根据种核的形态特征应用UPGMA法对120个银杏单株进行形态聚类。两种聚类方法的相关分析表明形态标记和ISSR标记所揭示的银杏古树的遗传关系未达显著水平。
(4)从9条ISSR引物中,挑选出扩增谱带较多的6条引物,对10株银杏古树共100粒种子的胚乳DNA进行ISSR分析,揭示银杏在个体水平上的遗传变异丰富度。试验共扩增出97条ISSR谱带,单倍体水平和二倍体水平统计的多态性条带分别为90条和39条,多态性条带百分率分别为92.78%和40.21%。检测出的平均杂合位点有55个,平均杂合率为56.91%,杂合率最高达到68.04%,最低也有45.36%,显示出该地银杏杂合水平都比较高。10株银杏个体的平均变异为H_E=0.203,H_o=0.300。四种种核类型间的遗传分化值为G_(ST)=0.137、φ_(ST)=16.71%。
(5)根据6条引物的扩增结果,对10个银杏单株分别从单倍体水平和二倍体水平进行分子聚类分析,得到相似但不完全相同的聚类图,基于单倍体水平的聚类比二倍体更为精确,所以得到的结果更为可靠。
Ginkgo biloba L.,belonging to Ginkgoaceae and Ginkgo,is one of the oldest remained tree species in gymnosperm and known as "living fossil".China is the native land of Ginkgo biloba and there are abundant ancient Ginkgo tree resources.Suizhou and Anlu of Hubei Province are two of the main concentrated distribution regions of ancient ginkgo trees in China.In this paper,based on of Fuhuimin's recearches about seed nucleus' characteristics and nutritional components' content of the ancient ginkgo trees in Suizhou and Anlu,the genetic variation of the ancient ginkgo trees here were studied by using ISSR molecular markers.Major results were as follows:
(1) A total of 145 bands were amplified using 9 ISSR primers to the genome DNA of those 120 Ginkgo seed samples.Of these bands,116 were polymorphic,and the percentage of polymorphic bands(PPB) was 80.00%.At the total level,Nei's gene diversity(H_E) was 0.269,Shannon's information index was 0.401.It showed that seeds of ginkgo trees here had high genetic variation.
(2) Percentage of polymorphic bands(PPB),observed number of alleles per locus (A_O),effective number of alleles per locus(A_E),Nei's gene diversity(H_E) and Shannon's information index(H_O) were used to reveal genetic variation within the 4 types populations.It showed that the variation extent decresased in sequence of Meihe type＞Maling type＞Yuanzi type＞Fozhi type.Among the four types,G_(ST) was 0.228,φST was 2.46%.This showed that the genetic variation was mainly distributed within types.
(3) UPGMA cluster of 120 individuals was made based on Genetic similarity(GS). UPGMA cluster of these trees was also made based on seed nucleus' characteristics. Correlation analysis between two kinds of cluster showed that there was no significant correlation between morphological markers and ISSR molecular markers.
(4) 6 ISSR primers which could amplify more bands were screened out from the 9 polymorphic ISSR primers.These effective primers were used to analyze the genetic variation of 100 seed samples of 10 ancient Ginkgo trees at individual level.As a result, 97 bands were generated,of which 90 bands were polymorphic based on haploid data and 39 bands were polymorphic based on diploid data,the percentage of polymorphism bands were 92.78%and 40.21%respectively.Heterozygosity test showed that the average number of heterozygous loci was 55 and the average heterozygosity was 56.91%.The highest heterozygosity was 68.04%,and the lowest was 45.36%.These results indicated that high heterozygosity existed in the Ginkgo trees in Suizhou and Anlu.Genetic variation at individual level was:PPB=55.16%,HE=0.203,H_O=0.300.Among the 4 seed nucleus types,G_(ST) was 0.139 andφST was 16.71%.
(5) According to the amplification results with the 6 ISSR primers to the 10 Ginkgo individuals,similar but not exactly the same dendrograms were generated through molecular genetic clustering at haploid level and diploid level.The duster based on haploid level was more accurate than that based on diploid level,so the former was more credible than the latter.
(1)用9条ISSR引物对120个银杏单株种核胚乳的基因组DNA进行PCR扩增,共得到145条清晰的扩增谱带。其中,多态性谱带116条,多态位点百分率(PPB)为80.00%。在总体水平上,Nei's基因多样性H_E为0.269,Shannon's多态性信息指数H_o为0.401,表明随州和安陆银杏种核在总体水平上具有较广泛的遗传变异。
(2)用多态位点百分率(PPB)、观测等位基因数(A_o)、有效等位基因数(A_E)、Nei's基因多样性值(H_E)以及Shannon's多态性信息指数(H_o)来度量四种种核类型内的遗传变异,结果表明:不同种核类型遗传变异程度依次是梅核类＞马铃类＞圆子类＞佛指类。四种种核类型间的遗传分化值G_(ST)为0.228,φ_(ST)=2.46%,说明银杏古树的遗传变异主要分布在类型内。
(3)根据单株间的遗传相似系数(GS),应用UPGMA法构建120个银杏单株的分子聚类图;根据种核的形态特征应用UPGMA法对120个银杏单株进行形态聚类。两种聚类方法的相关分析表明形态标记和ISSR标记所揭示的银杏古树的遗传关系未达显著水平。
(4)从9条ISSR引物中,挑选出扩增谱带较多的6条引物,对10株银杏古树共100粒种子的胚乳DNA进行ISSR分析,揭示银杏在个体水平上的遗传变异丰富度。试验共扩增出97条ISSR谱带,单倍体水平和二倍体水平统计的多态性条带分别为90条和39条,多态性条带百分率分别为92.78%和40.21%。检测出的平均杂合位点有55个,平均杂合率为56.91%,杂合率最高达到68.04%,最低也有45.36%,显示出该地银杏杂合水平都比较高。10株银杏个体的平均变异为H_E=0.203,H_o=0.300。四种种核类型间的遗传分化值为G_(ST)=0.137、φ_(ST)=16.71%。
(5)根据6条引物的扩增结果,对10个银杏单株分别从单倍体水平和二倍体水平进行分子聚类分析,得到相似但不完全相同的聚类图,基于单倍体水平的聚类比二倍体更为精确,所以得到的结果更为可靠。
Ginkgo biloba L.,belonging to Ginkgoaceae and Ginkgo,is one of the oldest remained tree species in gymnosperm and known as "living fossil".China is the native land of Ginkgo biloba and there are abundant ancient Ginkgo tree resources.Suizhou and Anlu of Hubei Province are two of the main concentrated distribution regions of ancient ginkgo trees in China.In this paper,based on of Fuhuimin's recearches about seed nucleus' characteristics and nutritional components' content of the ancient ginkgo trees in Suizhou and Anlu,the genetic variation of the ancient ginkgo trees here were studied by using ISSR molecular markers.Major results were as follows:
(1) A total of 145 bands were amplified using 9 ISSR primers to the genome DNA of those 120 Ginkgo seed samples.Of these bands,116 were polymorphic,and the percentage of polymorphic bands(PPB) was 80.00%.At the total level,Nei's gene diversity(H_E) was 0.269,Shannon's information index was 0.401.It showed that seeds of ginkgo trees here had high genetic variation.
(2) Percentage of polymorphic bands(PPB),observed number of alleles per locus (A_O),effective number of alleles per locus(A_E),Nei's gene diversity(H_E) and Shannon's information index(H_O) were used to reveal genetic variation within the 4 types populations.It showed that the variation extent decresased in sequence of Meihe type＞Maling type＞Yuanzi type＞Fozhi type.Among the four types,G_(ST) was 0.228,φST was 2.46%.This showed that the genetic variation was mainly distributed within types.
(3) UPGMA cluster of 120 individuals was made based on Genetic similarity(GS). UPGMA cluster of these trees was also made based on seed nucleus' characteristics. Correlation analysis between two kinds of cluster showed that there was no significant correlation between morphological markers and ISSR molecular markers.
(4) 6 ISSR primers which could amplify more bands were screened out from the 9 polymorphic ISSR primers.These effective primers were used to analyze the genetic variation of 100 seed samples of 10 ancient Ginkgo trees at individual level.As a result, 97 bands were generated,of which 90 bands were polymorphic based on haploid data and 39 bands were polymorphic based on diploid data,the percentage of polymorphism bands were 92.78%and 40.21%respectively.Heterozygosity test showed that the average number of heterozygous loci was 55 and the average heterozygosity was 56.91%.The highest heterozygosity was 68.04%,and the lowest was 45.36%.These results indicated that high heterozygosity existed in the Ginkgo trees in Suizhou and Anlu.Genetic variation at individual level was:PPB=55.16%,HE=0.203,H_O=0.300.Among the 4 seed nucleus types,G_(ST) was 0.139 andφST was 16.71%.
(5) According to the amplification results with the 6 ISSR primers to the 10 Ginkgo individuals,similar but not exactly the same dendrograms were generated through molecular genetic clustering at haploid level and diploid level.The duster based on haploid level was more accurate than that based on diploid level,so the former was more credible than the latter.
引文
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